Non-enzymic glycation of human extracellular superoxide dismutase.
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ABSTRACT:
SUBMITTER: Adachi T
PROVIDER: S-EPMC1151575 | biostudies-other | 1991 Oct
REPOSITORIES: biostudies-other
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Project description:The secretory tetrameric extracellular superoxide dismutase (EC-SOD) is the only glycosylated SOD isoenzyme. The importance of the carbohydrate moiety for the properties of the enzyme is unknown. An expression vector defining nonglycosylated EC-SOD (ngEC-SOD) was constructed by mutagenesis of the codon for Asn-89 into a codon for Gln. The vector was transfected into Chinese hamster ovary DXB-11 cells and ngEC-SOD was isolated to 70% purity from the culture media of selected clones. The absence of glycosylation was established by the lack of affinity for various lectins, the absence of staining with the periodic acid-Schiff reagent, the change in mobility and composition of the tryptic peptide containing the mutated glycosylation site, and the reduction in apparent molecular mass upon SDS/PAGE and size-exclusion chromatography. The tetrameric state was retained. The heparin affinity, a fundamental and distinguishing property of EC-SOD, was found to be slightly increased. The enzymic activity was essentially retained. The major difference from native glycosylated enzyme in physical properties was a marked reduction in solubility. Like glycosylated EC-SOD, ngEC-SOD was, after intravenous injection into rabbits, rapidly sequestered by the vessel endothelium, and was promptly released into plasma after injection of heparin. The only difference from glycosylated EC-SOD in this behaviour, was a slightly more rapid elimination of the mutant enzyme from the vasculature. It is concluded that no specific biological role for the EC-SOD carbohydrate moiety could be revealed.
| S-EPMC1132032 | biostudies-other
Project description:An important issue in critical care medicine is the identification of ways to protect the lungs from oxygen toxicity and reduce systemic oxidative stress in conditions requiring mechanical ventilation and high levels of oxygen. One way to prevent oxygen toxicity is to augment antioxidant enzyme activity in the respiratory system. The current study investigated the ability of aerosolized extracellular superoxide dismutase (EC-SOD) to protect the lungs from hyperoxic injury. Recombinant human EC-SOD (rhEC-SOD) was produced from a synthetic cassette constructed in the methylotrophic yeast Pichia pastoris. Female CD-1 mice were exposed in hyperoxia (FiO2>95%) to induce lung injury. The therapeutic effects of EC-SOD and copper-zinc SOD (CuZn-SOD) via an aerosol delivery system for lung injury and systemic oxidative stress at 24, 48, 72 and 96 h of hyperoxia were measured by bronchoalveolar lavage, wet/dry ratio, lung histology, and 8-oxo-2'-deoxyguanosine (8-oxo-dG) in lung and liver tissues. After exposure to hyperoxia, the wet/dry weight ratio remained stable before day 2 but increased significantly after day 3. The levels of oxidative biomarker 8-oxo-dG in the lung and liver were significantly decreased on day 2 (P<0.01) but the marker in the liver increased abruptly after day 3 of hyperoxia when the mortality increased. Treatment with aerosolized rhEC-SOD increased the survival rate at day 3 under hyperoxia to 95.8%, which was significantly higher than that of the control group (57.1%), albumin treated group (33.3%), and CuZn-SOD treated group (75%). The protective effects of EC-SOD against hyperoxia were further confirmed by reduced lung edema and systemic oxidative stress. Aerosolized EC-SOD protected mice against oxygen toxicity and reduced mortality in a hyperoxic model. The results encourage the use of an aerosol therapy with EC-SOD in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure, including acute respiratory distress syndrome (ARDS).
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Project description:Disruption of the oxidant/antioxidant balance in the lung is thought to be a key step in the development of many airway pathologies. Hence, antioxidant enzymes play key roles in controlling or preventing pulmonary diseases related to oxidative stress. The superoxide dismutases (SOD) are a family of enzymes that play a pivotal role protecting tissues from damage by oxidant stress by scavenging superoxide anion, which prevents the formation of other more potent oxidants such as peroxynitrite and hydroxyl radical. Extracellular SOD (EC-SOD) is found predominantly in the extracellular matrix of tissues and is ideally situated to prevent cell and tissue damage initiated by extracellularly produced ROS. EC-SOD has been shown to be protective in several models of interstitial lung disease, including pulmonary fibrosis. In addition, alterations in EC-SOD expression are also present in human idiopathic pulmonary fibrosis (IPF). This review discusses EC-SOD regulation in response to pulmonary fibrosis in animals and humans and reviews possible mechanisms by which EC-SOD may protect against fibrosis.
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Project description:A further characterization of human extracellular superoxide dismutase is reported. The study was especially aimed at the interaction with substances known to interfere with CuZn superoxide dismutase and other superoxide dismutases. Extracellular superoxide dismutase is efficiently inhibited by cyanide and is about 3 times more sensitive than is human CuZn superoxide dismutase. The sensitivity to azide is much lower, but still about 3 times higher than that of human CuZn superoxide dismutase. Extracellular superoxide dismutase is about as rapidly inactivated by hydrogen peroxide as is CuZn superoxide dismutase. The sensitivity to diethyldithiocarbamate is very high and more than an order of magnitude larger than that of CuZn superoxide dismutase. Sodium dodecyl sulphate, under conditions suggested as being suitable for distinguishing between the insensitive CuZn superoxide dismutase and the sensitive Mn superoxide dismutase, efficiently inactivated extracellular superoxide dismutase. No antigenic similarities between extracellular superoxide dismutase and CuZn superoxide dismutase could be demonstrated. Anti-(extracellular superoxide dismutase) did not bind CuZn superoxide dismutase, and anti-(CuZn superoxide dismutase) did not bind extracellular superoxide dismutase.
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Project description:To determine the effect of oxidative stress on expression of extracellular superoxide dismutase (EC-SOD), CuZn-SOD and Mn-SOD, two fibroblast lines were exposed for periods of up to 4 days to a wide concentration range of oxidizing agents: xanthine oxidase plus hypoxanthine, paraquat, pyrogallol, alpha-naphthoflavone, hydroquinone, catechol, Fe2+ ions, Cu2+ ions, buthionine sulphoximine, diethylmaleate, t-butyl hydroperoxide, cumene hydroperoxide, selenite, citiolone and high oxygen partial pressure. The cell lines were cultured both under serum starvation and at a serum concentration that permitted growth. Under no condition was there any evidence of EC-SOD induction. Instead, the agents uniformly, dose-dependently and continuously reduced EC-SOD expression. We interpret the effect to be due to toxicity. Enhancement of the protection against oxidative stress by addition of CuZn-SOD, catalase and low concentrations of selenite did not influence the expression of any of the SOD isoenzymes. Removal of EC-SOD from cell surfaces by heparin also did not influence SOD expression. Mn-SOD was moderately induced by high doses of the first 11 oxidants. Apart from reduction at high toxic doses, there were no significant effects on the CuZn-SOD activity by any of the treatments. Thus EC-SOD, previously shown to be profoundly influenced by inflammatory cytokines, was not induced by its substrate or other oxidants. In a similar fashion, Mn-SOD, previously shown to be greatly induced and depressed by cytokines, was only moderately influenced by oxidants. We suggest that the regulation of these SOD isoenzymes in mammalian tissues primarily occurs in a manner co-ordinated by cytokines, rather than as a response of individual cells to oxidants.
| S-EPMC1137946 | biostudies-other
Project description:Previous studies indicate that superoxide is important in the modulation of blood pressure but have not specifically identified the cell types or organs involved. We created mice with loxP sites flanking the extracellular superoxide dismutase (SOD3) gene. These mice were crossed with mice expressing inducible Cre-recombinase driven by the smooth muscle myosin heavy chain promoter allowing tissue-specific deletion of SOD3. Deletion of SOD3 increased vascular superoxide and reduced vascular NO levels as detected by electron spin resonance. Despite these changes in NO and superoxide, we did not observe increases in vascular inflammation caused by angiotensin II. Moreover, deletion of vascular SOD3 did not augment hypertension in response to angiotensin II. In additional studies, we also deleted SOD3 from the circumventricular organs by intracerebroventricular injection of an adenovirus encoding Cre-recombinase. Although this raised blood pressure and augmented the hypertension caused by angiotensin II, these responses were not further increased by vascular deletion of SOD3. These data suggest that the extracellular superoxide dismutase in vascular smooth muscle is not involved in the genesis of angiotensin II-induced hypertension and further emphasize the role of central SOD3 in the modulation of blood pressure.
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Project description:Reactive oxygen species (ROS) are increasingly recognized as critical determinants of cellular signaling and a strict balance of ROS levels must be maintained to ensure proper cellular function and survival. Notably, ROS is increased in cancer cells. The superoxide dismutase family plays an essential physiological role in mitigating deleterious effects of ROS. Due to the compartmentalization of ROS signaling, EcSOD, the only superoxide dismutase in the extracellular space, has unique characteristics and functions in cellular signal transduction. In comparison to the other two intracellular SODs, EcSOD is a relatively new comer in terms of its tumor suppressive role in cancer and the mechanisms involved are less well understood. Nevertheless, the degree of differential expression of this extracellular antioxidant in cancer versus normal cells/tissues is more pronounced and prevalent than the other SODs. A significant association of low EcSOD expression with reduced cancer patient survival further suggests that loss of extracellular redox regulation promotes a conducive microenvironment that favors cancer progression. The vast array of mechanisms reported in mediating deregulation of EcSOD expression, function, and cellular distribution also supports that loss of this extracellular antioxidant provides a selective advantage to cancer cells. Moreover, overexpression of EcSOD inhibits tumor growth and metastasis, indicating a role as a tumor suppressor. This review focuses on the current understanding of the mechanisms of deregulation and tumor suppressive function of EcSOD in cancer.
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Project description:Extracellular superoxide dismutase (EC-SOD), also known as SOD3, is an antioxidant expressed at high levels in normal adult kidneys. Because oxidative stress contributes to a variety of kidney injuries, we hypothesized that EC-SOD may be protective in CKD progression. To study this hypothesis, we used a murine model of ADR nephropathy characterized by albuminuria and renal dysfunction. We found that levels of EC-SOD diminished throughout the course of disease progression and were associated with increased levels of NADPH oxidase and oxidative stress markers. EC-SOD null mice were sensitized to ADR injury, as evidenced by increases in albuminuria, serum creatinine, histologic damage, and oxidative stress. The absence of EC-SOD led to increased levels of NADPH oxidase and an increase in β-catenin signaling, which has been shown to be pathologic in a variety of kidney injuries. Exposure of EC-SOD null mice to either chronic angiotensin II infusion or to daily albumin injections also caused increased proteinuria. In contrast, EC-SOD null mice subjected to nonproteinuric CKD induced by unilateral ureteral obstruction exhibited no differences compared with wild-type mice. Finally, we also found a decrease in EC-SOD in human CKD biopsy samples, similar to our findings in mice. Therefore, we conclude that EC-SOD is protective in CKDs characterized by proteinuria.
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Project description:Extracellular superoxide dismutase (EC-SOD) is the major SOD isoenzyme in extracellular fluids, but occurs also in tissues. The sites and characteristics of the synthesis of the enzyme are unknown. The occurrence of EC-SOD in cultures of a large panel of human cell lines was assayed by means of an e.l.i.s.a. Unlike the situation for the intracellular isoenzymes CuZn-SOD and Mn-SOD, expression of EC-SOD occurs in only a few cell types. None of the ten investigated suspension-growing cell lines produced EC-SOD. Among normal diploid anchorage-dependent cell lines, expression was found in all 25 investigated fibroblast cell lines, in the two glia-cell lines, but not in six endothelial-cell lines, two epithelial-cell lines or in two amnion-derived lines. Among neoplastic anchorage-dependent cell lines expression was found in 13 out of 29. EC-SOD was secreted into the culture medium by cell lines expressing the enzyme. The rate of EC-SOD synthesis varied by nearly 100-fold among the fibroblast lines and remained essentially constant in the individual lines during long-term culture. In the nine investigated cases, the secreted EC-SOD was of the high-heparin-affinity C type. It is suggested that tissue EC-SOD is secreted by a few well-dispersed cell types, such as fibroblasts and glia cells, to diffuse subsequently around and reversibly bind to heparan sulphate proteoglycan ligands in the glycocalyx of the surface of most tissue cell types and in the interstitial matrix.
| S-EPMC1131117 | biostudies-other
Project description:Polymorphisms in Superoxide dismutase 3, extracellular (SOD3) have been associated with reduced lung function and susceptibility to chronic obstructive pulmonary disease (COPD) in adults. Previously, we identified SOD3 as a contributing factor to altered ventilation efficiency (dead space volume/total lung capacity) in mice. Because SOD3 protects the extracellular matrix of the lung, we hypothesized that SOD3 variants also may influence postnatal lung function development. In this study, SOD3 transcript and protein localization were examined in mouse strains with differing ventilation efficiency [C3H/HeJ (high), JF1/Msf (low)] during postnatal lung development. Compared with C3H/HeJ mice, JF1/Msf mice had Sod3 promoter single nucleotide polymorphisms (SNPs) that could affect transcription factor binding sites and a decline in total lung SOD3 mRNA during postnatal development. In adult JF1/Msf mice, total lung SOD3 activity as well as SOD3 transcript and protein in airway epithelial and alveolar type II cells and the associated matrix decreased. In children (n = 1,555; age 9-11 yr), two common SOD3 SNPs, one located in the promoter region [C/T affecting a predicted aryl hydrocarbon receptor-xenobiotic response element (AhR-XRE) binding motif] and the other in exon 2 (Thr/Ala missense mutation), were associated with decreased forced expiratory volume in 1 s (FEV(1)), and the promoter SNP was associated with decreased maximal expiratory flow at 25% volume (MEF(25)). In vitro, a SOD3 promoter region-derived oligonucleotide containing the C variant was more effective in competing with the nuclear protein-binding capacity of a labeled probe than that containing the T variant. Along with the previous associated risk of lung function decline in COPD, these findings support a possible role of SOD3 variants in determining lung function in children.
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